Ex Situ Dual Hypothermic Oxygenated Machine Perfusion for Human Split Liver Transplantation

Abstract

Liver splitting allows the opportunity to share a deceased graft between 2 recipients but remains underutilized. We hypothesized that liver splitting during continuous dual hypothermic oxygenated machine perfusion (DHOPE) is feasible, with shortened total cold ischemia times and improved logistics. Here, we describe a left lateral segment (LLS) and extended right lobe (ERL) liver split procedure during continuous DHOPE preservation with subsequent transplantation at 2 different centers.

Methods:
After transport using static cold storage, a 51-year-old brain death donor liver underwent end-ischemic DHOPE. During DHOPE, the donor liver was maintained <10 °C and oxygenated with a Po2 of >106 kPa. An ex situ ERL/LLS split was performed with continuing DHOPE throughout the procedure to avoid additional ischemia time.

Results:
Total cold ischemia times for the LLS and ERL were 205 minutes and 468 minutes, respectively. Both partial grafts were successfully transplanted at 2 different transplant centers. Peak aspartate aminotransferase and alanine aminotransferase were 172 IU/L and 107 IU/L for the LLS graft, and 839 IU/L and 502 IU/L for the ERL graft, respectively. The recipient of the LLS experienced an episode of acute cellular rejection. The ERL transplantation was complicated by severe acute pancreatitis with jejunum perforation requiring percutaneous drainage and acute cellular rejection. No device-related adverse events were observed.

Conclusions:
Liver splitting during continuous DHOPE preservation is feasible, has the potential to substantially shorten cold ischemia time and may optimize transplant logistics. Therefore liver splitting with DHOPE can potentially improve utilization of split liver transplantation.

Full text

Transplantation DIRECT 2021 www.transplantationdirect.com 1
ISSN: 2373-8731
DOI: 10.1097/TXD.0000000000001116
Received 30 October 2020.
Accepted 16 November 2020.
1 Department of Surgery, Section of HPB Surgery & Liver Transplantation,
University of Groningen, University Medical Center Groningen, Groningen, The
Netherlands.
2 Department of Surgery, Division of HPB and Transplant Surgery, Erasmus MC
University Medical Center, Rotterdam, The Netherlands.
The authors declare no funding or conflicts of interest.
A.M.T., H.H., W.G.P., R.J.P., and V.E.d.M. participated in writing of the
article. A.M.T. and V.E.d.M. participated in data analysis. R.J.P. and V.E.d.M.
participated in study design. All authors participated in performance of the study.
All authors have read the article, contributed with critical revisions, and have
approved the final draft.
Supplemental digital content (SDC) is available for this article. Direct URL citations
appear in the printed text, and links to the digital files are provided in the HTML
text of this article on the journal’s Web site (www.transplantationdirect.com).
Correspondence: Vincent E. de Meijer, MD, PhD, Department of Surgery,
Liver Transplantation and HPB Surgery, University Medical Center Groningen,
Hanzeplein 1, 9713 GZ Groningen, The Netherlands. (v.e.de.meijer@umcg.nl).
Copyright © 2021 The Author(s). Transplantation Direct. Published by Wolters
Kluwer Health, Inc. This is an open-access article distributed under the terms of
the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0
(CCBY-NC-ND), where it is permissible to download and share the work provided
it is properly cited. The work cannot be changed in any way or used commercially
without permission from the journal.
Ex Situ Dual Hypothermic Oxygenated Machine
Perfusion for Human Split Liver Transplantation
Adam M. Thorne, BSc,1 Veerle Lantinga, BSc,1 Silke Bodewes, BSc,1 Ruben H. J. de Kleine, MD,1
Maarten W. Nijkamp, MD, PhD,1 Joost Sprakel, MD,1 Hermien Hartog, MD, PhD,2 Wojciech G. Polak, MD, PhD,2
Robert J. Porte, MD, PhD,1 and Vincent E. de Meijer, MD, PhD1
With an ongoing disparity between supply and demand
for transplantable livers, particularly for pediatric
recipients, it has become increasingly important to nd new
methods of both expanding the donor pool and improving
graft quality.1 One method for increasing the number of
available grafts is to split the deceased donor liver and share
the graft between 2 recipients.2 For the majority of pediatric
recipients, a left lateral segment (LLS) split sufces, while the
extended right lobe (ERL) can be transplanted into an adult
recipient. Liver splitting typically takes place on the back table
with the graft immersed in ice-cold preservation solution. The
splitting procedure itself, as well as subsequent transport of
the ERL to a remote transplant center prolongs cold ischemia
time (CIT), which may negatively affect patient outcome after
transplantation.3 As a solution, in situ liver splitting, similar to
that seen in living donor procedures, was developed as a way
to reduce CITs.4 This, however, prolongs operation time dur-
ing organ retrieval and may complicate logistics.
End-ischemic ex situ hypothermic oxygenated machine per-
fusion has seen increasing utilization in recent years due to its
ability to mitigate ischemia/reperfusion injury (IRI) in donation
after circulatory death liver transplantation.5 This method can
be applied to single hypothermic machine perfusion through
the portal vein (HOPE) or dual perfusion through the portal
vein and hepatic artery (dual hypothermic oxygenated machine
perfusion [DHOPE]). The application of end-ischemic dynamic
machine preservation by DHOPE during split liver procedures
could provide an interesting strategy to reduce prolonged CITs
associated with ex situ liver splitting. Furthermore, replenish-
ment of ATP during DHOPE and maintaining a constantly sta-
ble temperature during the split may attenuate IRI, improving
Liver Transplantation
Background. Liver splitting allows the opportunity to share a deceased graft between 2 recipients but remains underutilized.
We hypothesized that liver splitting during continuous dual hypothermic oxygenated machine perfusion (DHOPE) is feasible, with
shortened total cold ischemia times and improved logistics. Here, we describe a left lateral segment (LLS) and extended right
lobe (ERL) liver split procedure during continuous DHOPE preservation with subsequent transplantation at 2 different centers.
Methods. After transport using static cold storage, a 51-year-old brain death donor liver underwent end-ischemic DHOPE.
During DHOPE, the donor liver was maintained <10 °C and oxygenated with a Po2 of >106 kPa. An ex situ ERL/LLS split was
performed with continuing DHOPE throughout the procedure to avoid additional ischemia time. Results. Total cold ischemia
times for the LLS and ERL were 205 minutes and 468 minutes, respectively. Both partial grafts were successfully transplanted
at 2 different transplant centers. Peak aspartate aminotransferase and alanine aminotransferase were 172 IU/L and 107 IU/L
for the LLS graft, and 839 IU/L and 502 IU/L for the ERL graft, respectively. The recipient of the LLS experienced an episode of
acute cellular rejection. The ERL transplantation was complicated by severe acute pancreatitis with jejunum perforation requiring
percutaneous drainage and acute cellular rejection. No device-related adverse events were observed. Conclusions. Liver
splitting during continuous DHOPE preservation is feasible, has the potential to substantially shorten cold ischemia time and may
optimize transplant logistics. Therefore liver splitting with DHOPE can potentially improve utilization of split liver transplantation.
(Transplantation Direct 2021;7: e666; doi: 10.1097/TXD.0000000000001116. Published online 4 February, 2021.)

2 Transplantation DIRECT ■ 2021 www.transplantationdirect.com
organ quality and outcome in both partial grafts.6 The combi-
nation of above-mentioned advantages may feasibly improve
transplant logistics.
Here, we present a case report demonstrating the technical
aspects of liver splitting during dynamic machine preservation
with DHOPE, after which both partial grafts were success-
fully transplanted at 2 different centers.
MATERIALS AND METHODS
DHOPE is implemented as standard practice in our center
for donation after circulatory death liver transplantation
and can be applied for logistical reasons such as expected
prolonged CITs (eg, in case of retransplantation or ex situ
split). No formal medical ethical committee approval was
obtained for this case. The Declaration of Helsinki and the
Declaration of Istanbul were adhered to.
A liver graft was accepted from a 51-year-old brain death
donor in a regional hospital who suffered from cerebrovascular
bleeding. The donor weight was 70 kg, height 192 cm, and had
a calculated body mass index of 19 kg/m2. The Eurotransplant
donor risk index was 1.62. Organ procurement was performed in
a standard fashion. During procurement, the donor organs were
ushed via the cannulated aorta using 5 liters of cold, heparin-
ized (25 000 IU) modied University of Wisconsin (UW) preser-
vation solution. Hepatectomy was completed after 38 minutes
from start of cold perfusion, and after an additional portal back
table ush with 2 liters of UW solution, the liver was placed in
static cold storage (SCS) for transport to the splitting center. A
5 cm cylindrical segment of supratruncal aorta was left attached
to the celiac trunk during procurement for cannulation purposes.
Upon arrival at the splitting center (full timeline represented
in Figure1), the liver was immersed in ice-cold UW solution
for back table procurement to prepare for dual cannulation
allowing DHOPE preservation using the portal vein for portal
perfusion and the supratruncal aorta for arterial perfusion, as
described previously.7 In brief, the liver was placed in supine
position in the reservoir of a LiverAssist device (Organ Assist,
Groningen, The Netherlands), after which the 24F portal vein
and hepatic artery cannulas were subsequently connected to
the perfusion system. A continuous portal ow was provided
with a pressure of 3 mm Hg. Hepatic artery pressure was
set and maintained at 25 mm Hg with pulsatile ow of 60/
min throughout the perfusion. Temperature was maintained
at <10 °C throughout the perfusion. The perfusion solution
comprised 4 L UW machine perfusion solution (PumpProtect;
Carnamedica, Warsaw, Poland) and was oxygenated (100%
oxygen at 1 L/min) with a P2 of >106 kPa.
Portal venous and hepatic arterial ow and pressure
parameters were maintained and recorded every 15 minutes.
Perfusate analysis was performed every 30 minutes using an
ABL90 FLEX blood gas analyzer (Radiometer, Denmark).
Ex situ LLS and ERL split was performed in the LiverAssist
reservoir during continuous DHOPE throughout the procedure
(Figure2A–D) by 2 surgeons assisted by a surgical nurse (Video,
SDC, http://links.lww.com/TXD/A308). Surgeon 1, standing at
the front of the LiverAssist, used the Cavitron ultrasonic sur-
gical aspirator device (Excel+; Integra LifeSciences, Tullamore,
Ireland), with simultaneous ligation and cutting of exposed
microvasculature/bile ducts in the parenchymal transection
plane performed by surgeon 2, standing at the back of the
LiverAssist. A gauze was placed underneath the liver to prevent
any Cavitron ultrasonic surgical aspirator-related tissue debris
from entering the perfusion system and preclude potential
obstruction of the oxygenators. Additionally, a piece of silicone
tubing was placed underneath the transection plane and con-
nected to the rim of the reservoir to establish a reversed hanging
maneuver, “folding” the graft open along the transection plane
like a book, allowing for improved visualization. The left hepatic
vein was separated from the middle hepatic vein and caval vein,
and good venous outow from both LLS and ERL was observed.
After parenchymal transection, both LLS and ERL remained
adequately perfused via the portal and arterial branches, indi-
cated by stable perfusion parameters (Figure2A–E). Finally, the
hilar plate (including the bile duct) was identied and divided
at the plane between S4 and S2/3, resulting in complete division
except for the hepatic arteries and portal veins. Because the LLS
was allocated to a pediatric recipient at the splitting center, the
timing of vascular division was performed in accordance with
the surgical team of the pediatric recipient to minimize the sec-
ond SCS time. When the recipient went anhepatic, the left portal
vein and left hepatic artery of the donor graft were divided and
the stump to the main portal vein and proper hepatic artery
were over sewn. The LLS was removed, immediately immersed
in ice-cold UW solution, and transferred to the recipient operat-
ing room, while the ERL remained in the reservoir with continu-
ing machine perfusion (Figure 2E). Subsequently, the ERL was
removed from the device, immediately immersed in ice-cold UW
solution, and packed in polystyrene box with ice for transporta-
tion to the second transplant center.
FIGURE 1. Timeline of dual hypothermic oxygenated machine perfusion (DHOPE) split liver procedure into the left lateral segment (LLS) and
extended right lobe (ERL). CIT, cold ischemia time; HA, hepatic artery; PV, portal vein; SCS, static cold storage.

© 2021 The Author(s). Published by Wolters Kluwer Health, Inc. Thorne et al 3
RESULTS
The rst CIT for the SCS-preserved organ was 174 minutes,
including 129 minutes of transport to our center, followed by
a back-table procedure and cannulation for another 45 min-
utes (Figure1).
During DHOPE, hepatic arterial and portal venous ow
rates were 50–60 mL/min at 25 mm Hg and 80–120 mL/min
at 3 mm Hg, respectively. Slight uctuations in ow occurred
due to manipulation of the liver during the split procedure.
DHOPE preservation time for the LLS was 125 minutes. The
FIGURE 2. The progression of the split procedure is observable from (A) start of dual hypothermic oxygenated machine perfusion (DHOPE),
(B) start of left lateral segment (LLS)/extended right lobe (ERL) liver split with division of the middle and left hepatic vein with magnification of the
transection plane, (C) midway through parenchymal liver split using the CUSA device, (D) demonstrating full parenchymal separation of the LLS
from the ERL, and (E) showing dual perfusion of the ERL only, after the LLS has been fully removed. CUSA, Cavitron ultrasonic surgical aspirator;
GB, gall bladder; HA, hepatic artery; PV, portal vein; TP, transection plane.

4 Transplantation DIRECT ■ 2021 www.transplantationdirect.com
ERL remained on the pump for a further 27 minutes while
the LLS was being prepared for implantation and transported
to the recipient operating room. The duration of the split-
ting procedure during DHOPE preservation was 110 minutes
(Figure1). Total preservation time for the LLS was 355 min-
utes. Postperfusion weight was 216 g for the LLS and 1082 g
for the ERL.
Implantation of the LLS and portal reperfusion required
46 minutes. The hepatic arterial anastomosis required an
additional 31 minutes, giving a total anastomosis time of 77
minutes. Perioperative blood loss was 3.4 liters. The recipi-
ent was supplemented with 5 units of red blood cells, 2 units
of plasma, 1.2 g brinogen, and 200 mg tranexamic acid. The
postoperative course was complicated by a reoperation for
removal of a hematoma at postoperative day 3 and a grade 2
(biopsy-proven) episode of acute cellular rejection treated by
high dose steroids after 27 days. LLS recipient initially expe-
rienced a peak-rise of both aspartate aminotransferase (172
IU/L) and alanine aminotransferase (107 IU/L), followed by a
decrease in the rst 4 days posttransplant. The second increase
from day 4 to beyond day 7 of both markers was most likely
related to the grade 2 acute cellular rejection (Figure3A). On
day 7, bilirubin was 2.46 mg/dL and international normalized
ratio was 1.3.
The total CIT for the ERL was 468 minutes, and a total
preservation time of 622 minutes (Figure 1). The ERL was
reperfused via the portal vein after 38 minutes and subse-
quently via the hepatic artery after 31 minutes. Perioperative
blood loss was 6.2 liters. The recipient was supplemented
with 4 units of red blood cells, 4 units of plasma, and 1 unit
of platelets. The postoperative course was complicated by
severe acute pancreatitis with jejunum perforation requiring
percutaneous drainage and acute cellular rejection (biopsy-
proven) treated with increased immunosuppression. Peak of
both aspartate aminotransferase (839 IU/L) and alanine ami-
notransferase (502 IU/L) in the ERL recipient was seen on
day 2 posttransplant followed by a decrease of transaminases
in the subsequent 5 days (Figure3B). On day 7, bilirubin was
12.2 mg/dL and international normalized ratio was 1.4.
At 6-month follow-up, both LLS and ERL recipients are at
home with good functioning grafts. No device-related adverse
events were observed during follow-up.
DISCUSSION
Liver splitting has the opportunity to expand 1 scarce
resource into 2, thereby adding great value in liver trans-
plantation for vulnerable recipients such as pediatrics and
small adults. Despite improvements in surgical techniques
and expertise, split liver transplantation remains underuti-
lized.8 Typically, split liver procedures take place on the back-
table under ischemic SCS conditions. One major advantage
of DHOPE is the protective mechanisms induced by keeping
the organ both cold and oxygenated during the split proce-
dure. This substantially shortens the ischemic SCS time. End-
ischemic DHOPE resuscitates mitochondria, leading to ATP
replenishment during dynamic preservation. Subsequently, the
production of reactive oxygen species after reperfusion in the
recipient is reduced, mitigating IRI.5
Another advantage of dual perfusion is that potential
variation in the arterial anatomy becomes more obvious.
Arteries are lled with pulsatile ow, leading to better visu-
alization. This also makes it easier to identify leaks from
arterial branches that may not have been ligated. A poten-
tial disadvantage is that the current LiverAssist device does
not allow performance of an intraoperative cholangiogram in
the context of bile duct division planning during perfusion.
However, if necessary, this can theoretically be accomplished
by extended tubing and using a radiolucent bowl.
At present, only 1 other case report of ex situ liver split-
ting with concurrent DHOPE exists, where a 19-year-old brain
death donor liver was split for implantation into 2 pediatric
recipients, with a hyperreduction of the LLS to S2 for trans-
plant to a neonate. The authors demonstrated positive results,
with mild IRI and no device-related adverse events.9 Both grafts
were transplanted at the same center, and therefore, no second
transportation was involved; however, they report a total CIT
of 11 and 14 hours for LLS and ERL, respectively. In our study,
DHOPE allowed for a substantial reduction in CIT, particu-
larly in the case of the ERL, where total CIT was reduced to
<8 hours even with the addition of a second transport time to
a second center (294 min transport and back table).
Liver splitting during normothermic machine perfusion
(NMP) has previously been demonstrated as a proof of con-
cept on human grafts rejected for transplant.10-12 These stud-
ies proposed splitting during NMP as a method of viability
FIGURE 3. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, and total lactate levels in the recipients
of (A) left lateral segment (LLS) and (B) extended right lobe (ERL) during the first 3 mo after transplantation. The increase in AST and ALT 5 d
posttransplant seen in the LLS graft recipient is reflective of an episode of (biopsy-proven) acute rejection.

© 2021 The Author(s). Published by Wolters Kluwer Health, Inc. Thorne et al 5
assessment, logistical improvement, and of potential benet
to the graft by reducing ischemia times. Although functional
assessment is not possible at hypothermic temperatures, in
optimal, high-quality grafts such as the 1 reported here, func-
tional assessment and viability testing are not necessary. Liver
splitting during NMP may add increased risk of injury through
additional and unnecessary rewarming steps, increasing warm
ischemia times. ERL grafts traveling to another recipient hos-
pital after splitting will also undergo an additional episode of
cooling, SCS, and rewarming. The effects of repeat cycles of
rewarming on liver grafts are unknown.
DHOPE has several advantages when compared with
NMP for liver splitting. Firstly, there is no recooling phase
between end of NMP and SCS for transport, and thus addi-
tional injury from temperature change is avoided. Second,
DHOPE poses a lower risk to the organ should there be a
technical issue with the perfusion machine. In the event of
such an issue, the graft is simply returned to SCS conditions
without the need for rapid cooling and ushing that would
be necessary during NMP. Furthermore, the liver is under
minimal metabolic demand during DHOPE preservation.
The split graft to be transplanted in the splitting center is in
optimal condition for implantation due to resuscitation from
end-ischemic DHOPE. The split graft traveling to a separate
transplant center is subjected to a second phase of CIT after
initial the split, however, does benet from a shorter ischemic
preservation time and from the oxygenated resuscitation dur-
ing the split procedure. This is preferable over end-ischemic
NMP, where the organ is not resuscitated before perfusion
at normothermia (37 °C). Finally, the combination of the
discussed advantages above may feasibly improve logistical
obstacles.
Our technique of vascular splitting at the level of the left
portal vein and left hepatic artery allowed continuing DHOPE
preservation of the ERL graft. There is currently no evidence
that HOPE is inferior to DHOPE, meaning that HOPE with
portal vein perfusion could be continued in cases where the
proper hepatic artery is used for the LLS. This, however, would
not be possible with NMP, as sufcient oxygenation of the bile
ducts via the hepatic artery is essential at 37 °C. Therefore, (D)
HOPE may facilitate sequential liver transplantation of the
ERL graft at the same center.9 Additionally, our report pro-
vides further evidence that DHOPE for split liver transplanta-
tion is feasible and can be an attractive therapeutic solution to
grafts with expected prolonged CIT.
We propose that the technique of liver splitting during con-
tinuous DHOPE has the potential to improve logistics and
utilization of split liver transplantation and could be a use-
ful strategy to shorten ischemic SCS time and mitigate sub-
sequent IRI.
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